Although this invention is not limited to cuvettes used for nucleic acid amplification, the background is described in the context of the latter.
Nucleic acid amplification generally proceeds via the following steps (shown in FIG. 1):
(1) If DNA is to be amplified, a complete DNA double helix is optionally chemically excised, using an appropriate restriction enzyme(s), to isolate the region of interest.
(2) A solution of the isolated nucleic acid portion (here, DNA) and nucleotides is heated to and maintained at 92.degree.-95.degree. C. for a length of time, e.g., no more than about 10 minutes, to denature the two nucleic acid strands: i.e., cause them to unwind and separate and form a template.
(3) The solution is then cooled through a 50.degree.-60.degree. C. zone to cause a primer nucleic acid strand to anneal or "attach" to each of the two template strands. To make sure this happens, the solution is held at an appropriate temperature, such as about 55.degree. C. for about 15 seconds, in an "incubation" zone.
(4) The solution is then heated to and held at about 70.degree. C., to cause an extension enzyme, preferably a thermally stable enzyme such as a polymerase isolated from thermus aquaticus, to extend the primer strand bound to the template strand, by using the nucleotides that are present.
(5) The completed new pair of strands is heated to 92.degree.-95.degree. C. again, for about 10-15 seconds, to cause this pair to separate.
(6) Steps (3)-(5) are then repeated, a number of times until the appropriate number of strands are obtained. (See, e.g., U.S. Pat. No. 4,683,202 for further illustration.) The more repetitions, the greater the number of multiples of the nucleic acid (here, DNA) that is produced. Preferably the desired concentration of nucleic acid is reached in a mininum amount of time.
A cuvette is usually used to hold the solution while it passes through the aforementioned temperature stages. Depending upon the design given to the cuvette, it can proceed more or less rapidly through the various stages. A key aspect controlling this is the thermal transfer efficiency of the cuvette--that is, its ability to transfer heat more or less instantaneously to or from all of the liquid solution within the cuvette. The disposition and the thermal resistance of the liquid solution itself are the major aspects affecting the thermal transfer, since portions of the liquid solution that are relatively far removed from the heat source or sink, will take longer to reach the desired temperature.
The crudest and earliest type of cuvette used in the prior art is a test tube, which has poor thermal transfer efficiency since (a) the walls of the cuvette by being glass or plastic, do not transfer thermal energy well, and (b) a cylinder of liquid has relatively poor thermal transfer throughout the liquid. That is, not only does the liquid have low thermal conductivity, but also a cylinder of liquid has a low surface to volume ratio, that is, about 27 in.sup.-1 for a fill of about 100 .mu.l.
Still another problem in DNA amplification is the manner in which the cuvette allows for ready removal of the liquid after reaction is complete. A test tube configuration readily permits such removal. However, modification of the cuvette to provide better thermal transfer efficiency tends to reduce the liquid transferability.
Recent cuvette or vessel constructions for reaction of liquid are shown in U.S. Pat. Nos. 4,426,451 issued Jan. 17, 1984 and 3,691,017 issued on Sept. 12, 1972. In the former, little attempt is made to provide high thermal transfer efficiency, except that the liquid is distributed as a thin film that will allow rapid heating, if heat penetrates to the liquid. However, no mention is made of the cuvette being constructed of metal or any material that is highly thermally conductive. Furthermore, since the spacing between top and bottom walls is no greater than 125 microns to provide a strong capillary effect, removal of liquid from such cuvette will be difficult. At best, not all the liquid will be removed because of the strong capillary attraction.
In the case of cuvettes of U.S. Pat. No. 3,691,017, more features suitable for DNA amplification are provided. For example, by having a spacing of about 5 mm between major surfaces 23 and 24, it is more readily possible to remove all of the liquid, there being less capillary attraction left at such a gross spacing. In addition, a metal layer 38A is provided on the outside of the cuvette to provide contact with a heating device. However, this cuvette does not have a low thermal time constant for several reasons. One reason is that to ensure transparency, the surfaces 23 and 24 are not constructed of metal, but rather of an insulator. As a result, a long thermal transfer path is needed by extending metal 38A around the edge of the device and into only a portion 35 of the volume of the cuvette. This thermal path length is well in excess of 0.5 mm since it is much more than the thickness of the wall providing the major surface 23 or 24. Indeed, only a portion of the volume of the cuvette is in direct contact with the metallic thermal-energy transfer element.
Secondly, and more importantly, there is a high thermal resistance in the cuvette of the '017 patent because of the low fluid surface/volume ratio provided by cavity geometry, discussed hereinafter in detail.